EP0730473B1 - Hirudin-konjugate aus hirudin und lipophilen verbindungen - Google Patents

Hirudin-konjugate aus hirudin und lipophilen verbindungen Download PDF

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EP0730473B1
EP0730473B1 EP95901432A EP95901432A EP0730473B1 EP 0730473 B1 EP0730473 B1 EP 0730473B1 EP 95901432 A EP95901432 A EP 95901432A EP 95901432 A EP95901432 A EP 95901432A EP 0730473 B1 EP0730473 B1 EP 0730473B1
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hirudin
acid
lipophilic
lipophilic compounds
conjugates
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EP0730473A1 (de
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Jürgen Schweden
Peter Eckes
Wilfried Hornberger
Thomas Subkowski
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Abbott GmbH and Co KG
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BASF SE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to new hirudin conjugates which consist of a Hirudin and lipophilic compounds are formed, the Production and their use as medicines.
  • Hirudin is a long-known, naturally occurring one Protein with anticoagulant properties. It is the strongest and most selective thrombin inhibitor known to date (Naturwissenschaften, 42, 537, (1955); Hoppe-Seylers Z. für Biol. Chemistry 366, 379 (1985)). Natural hirudin is a mixture of chemically related peptides with a molecular weight of 6900 - 7000 daltons and 64-66 amino acids. So far about 20 have been natural occurring hirudin variants are described (Scharf et al., FEBS-Letters 255, 105-110, (1989)).
  • EP 345616 describes coupling products made from hirudin and polymers Porters described. Soluble and insoluble become carriers Polymers such as dextran, sepharose, heparin, laevan and gelatin partial hydrolyzate called. The carrier-modified hirudins are said to improved pharmacological properties such as an extended one Possess half-life.
  • hirudin-polyalkylene glycol conjugates which is a cheaper one compared to natural hirudin pharmacological activity profile, such as an extended biological Efficacy and better bioavailability.
  • hirudin derivatives described with prolonged effectiveness by Modification of hirudin with a high molecular weight polymer is achieved.
  • the molecular weight of the polymer is a few Kilodalton, so the molecular weight of the polypeptide hirudin is significant is enlarged.
  • high molecular weight polymers are chemically not exactly defined; rather, they form one Population of different molecules that span a molecular weight range distributed.
  • Hirudin coupling products are not made from a single chemical Compound, but from molecular populations that relate to each other distinguish their molecular weight from each other.
  • hirudin conjugates formed from a hirudin and one or more lipophilic compounds, wherein the lipophilic compound has an octanol-water partition coefficient of more than 1.8 and chemically covalent with to the hirudin.
  • Such hirudins and their manufacture are, for example, from EP 171 024, EP 158 986, DE 38 055 406, WO 92/1712, GB 2 247 239, EP 557 199, WO 92/5748, DE 40 14 260 are known.
  • peptides or are structurally derived from hirudin Peptide derivatives with hirudin activity as described, for example, in EP 333 356 can be described, good for the production of the Hirudin conjugates according to the invention are suitable.
  • hirudins in which the attachment is used are preferred the lipophilic compound to no loss of activity leads. This is the case with hirudins, for example the linkage takes place via side chains of amino acids 27 to 37.
  • the amino acid positions are particularly advantageous for the attachment 27 and 33 (based on the natural hirudin HVI) because these connections are at the tip of a finger-like Region and the interaction of hirudin with thrombin is not to disturb.
  • the binding of the lipophilic compounds, optionally the Spacers with the lipophilic compounds can be attached to the amino groups of the Hirudins, for example the free N-terminal amino group, Amino groups of the lysine side chains, amino groups of the histidines, Amidine groups of arginines, or on hydroxyl groups of hirudin, for example of tyrosine, serine or threonine side chains respectively.
  • Particularly suitable for connecting the lipophilic compounds are the amino groups of the lysine side chains.
  • lipophilic compound it is also possible to bind the lipophilic compound to a modified tyrosine of hirudin, which is produced by nitration and Reduction is accessible (Meth. Enzymol. 25, 515-521, (1972)). On the arylaminotyrosine formed can then be the lipophilic compound can be linked with known coupling methods.
  • Suitable lipophilic compounds are compounds which have a or more functional groups such as amino, hydroxy, carboxy or have sulfonic acid groups and which have an octanol-water partition coefficient of greater than 1.8.
  • the lipophilic compounds can be natural products, e.g. saturated or unsaturated fatty acids, terpenes, prostaglandins, fat-soluble vitamins, carotenoids or steroids as well synthetic carboxylic acids, alcohols, amines and sulfonic acids with one or more alkyl, aryl, alkenyl or multiple unsaturated compounds that are both linear and branched can be and optionally with halogen, nitro, cyano, Alkoxy, alkylthio or haloalkyl groups are substituted.
  • natural products e.g. saturated or unsaturated fatty acids, terpenes, prostaglandins, fat-soluble vitamins, carotenoids or steroids as well synthetic carboxylic acids, alcohols, amines and sulfonic acids with one or more alkyl, aryl, alkenyl or multiple unsaturated compounds that are both linear and branched can be and optionally with halogen, nitro, cyano, Alkoxy, alkylthio
  • lipophilic compounds are the saturated fatty acids Caproic acid, caprylic acid, capric acid, lauric acid, Myristic acid, palmitic acid, stearic acid, arachic acid and Behenic acid and the unsaturated fatty acids palmitoleic acid, Oleic acid, linoleic acid, linolenic acid, ricinoleic acid, octadecatetraenoic acid, Eicosaenoic acid, eicosadienoic acid, arachidonic acid, eicosapentaenoic acid or erucic acid and the fatty alcohols obtainable from them and fatty amines.
  • Mixtures of fatty acids such as those found in are also suitable saponification of natural fats such as coconut oil, palm kernel oil, Rapeseed oil, olive oil, sunflower oil, high oleic sunflower oil, Castor oil or beef tallow.
  • lipophilic compounds are the carotenoids zeaxanthin, rhodovibrin or astaxanthin, the steroids cholesterol desmosterol, coprosterol, cerebrosterol, lathosterol, ergesterol, sitosterol, stigmasterol, cholanic acid, cholinic acid, dehydrocorticosterol, aldosterone test, lorosterol, lorosterol, andosterol, lorosterol, andosterol, lorosterol, lorosterol, andosterol, lorosterol, lorosterol, andosterol, lorosterol, andosterol, lorosterol, andosterol, lorosterol, androsterol Terpenes geraniol, nerol, linalool, menthol, carveol, borneol, farnesol, nerolidol or sclareol, the prostaglandins
  • Particularly preferred compounds of the formula I are those in which the radicals R 1 and R 2 consist of 4 to 16 carbon atoms and which are saturated or mono- or trisaturated.
  • the hirudin conjugates according to the invention are produced by using a hirudin either directly or using a spacer one or more lipophilic compounds is linked.
  • All bi- or multifunctional molecules are used as spacers suitable because of their multifunctional groups Linking - if necessary after activation - of hirudin and allow the lipophilic compound.
  • Particularly suitable spacers are amino acids, oligopeptides, mono-, di- or oligosaccharides, amino or hydroxycarboxylic acids, in particular those with a chain length of 2 to 10 carbon atoms, which can optionally carry further substituents to increase the hydrophilicity, for example C 1 - C 4 oligoalkylene glycols.
  • Suitable lipophilic compounds that have one or more functional Groups such as amino, hydroxy, carboxy or carry sulfonic acid groups because these are functional groups after activation with the reactive groups of hirudin can react.
  • the type of activation of the lipophilic compounds, if any the linkage via a spacer depends on the functional group and can be known in the art Way.
  • Carries the lipophilic compound or spacer with the lipophilic Connection a carboxyl group the activation can for example by conversion into an alkenyl ester, aryl ester, an O-hemiacetal, O-acyl half-amino acetal, O-acyl half-ketal, O-acyl half-aminoketal, O-acyl lactim, symmetrical or asymmetrical Carboxylic acid anhydride, carboxylic acid carbamic acid anhydride, O-acylisoureid, O-acyl-N-alkylhydroxylamine, an O-acylhydroxamic acid, an O-acyl-N, N-diacylhydroxylamine, O-acyloxime, O-acyl-N-azo-N-acylhydroxylamine, O-acyl-N-azo-N-arylhydroxylamine, carboxylic acid-sulfuric anhydride, Carboxylic acid-sulfuric anhydride, Carboxylic acid-phosphoric anhydr
  • Particularly suitable reactive groups are carboxylic acid chlorides, symmetrical anhydrides, optionally by 1, 2, 3 or 5 halogen or nitro groups substituted aryl esters, N-hydroxysuccinimide esters, N-hydroxyphthalimide ester or N-hydroxybenzotriazole ester.
  • Sulfonic acid groups can be activated in an analogous manner.
  • the second functional group is a carboxyl or Carries sulfonic acid group and in the manner described above for the coupling to the hirudin is activated.
  • Alkyldiketenes are also well suited as lipophilic compounds, which is linked directly to the hirudin without further activation can be.
  • Particularly preferred compounds of the general formula III are Fatty alkyl diketenes, which are described, for example, in DE 2927118 described, from the saturated fatty acids caproic acid, caprylic acid, Capric acid, lauric acid, myristic acid, palmitic acid, Stearic acid, arachic acid and behenic acid and the unsaturated Fatty acids palmitoleic acid, oleic acid, linoleic acid, linolenic acid, Ricinoleic acid, octadecatetraenoic acid, eicosaenoic acid, eicosadienoic acid, Arachidonic acid, eicosapentaenoic acid or erucic acid deduce.
  • fatty alkyl diketenes can also be used derived from mixtures of fatty acids, such as those used in saponification natural fats such as coconut fat, palm kernel fat, rapeseed oil, Olive oil, sunflower oil, high oleic sunflower oil, castor oil or beef tallow.
  • hirudin conjugates described according to the invention are chemical exactly defined and show a cheaper compared to hirudin pharmacological activity profile. Among other things, you own one significantly prolonged biological effectiveness and better Bioavailability. They also allow the hydrophobic Hirudine a targeting of the active ingredient on the surfaces of Blood cells and blood vessel surfaces.
  • the hirudin conjugates according to the invention valuable medicines for treatment and prophylaxis of thrombin-dependent thromboembolic events such as deep Venous thrombosis, pulmonary embolism, myocardial or cerebral infarction and unstable angina, continue to treat disseminated people Intravascular coagulation (DIC) and as a co-medication with thrombolytics such as streptokinase, urokinase, prourokinase, t-PA, APSAC, Plasminogen activators from the salivary glands of animals, as well the recombinant and mutant forms of all of these substances Shortening the reperfusion time and lengthening the reocclusion time.
  • DIC Intravascular coagulation
  • thrombolytics such as streptokinase, urokinase, prourokinase, t-PA, APSAC, Plasminogen activators from the salivary glands of animals, as well the
  • Another area of application is the prevention of thrombin-dependent early reocclusion and later restenosis after PTCA, preventing thrombin-induced proliferation smoother Muscle cells, preventing the accumulation of active thrombin in the CNS (e.g. in M. Alzheimer), tumor control and prevention of mechanisms leading to adhesion and metastasis from Lead tumor cells.
  • the new compounds can be used in the usual galenic Application forms can be used in solid or liquid form, e.g. as Solutions, ointments, creams or sprays. These are more common Manufactured way.
  • the active ingredients can with the usual pharmaceutical auxiliaries such as fillers, preservatives, Flow regulators, wetting agents, dispersants, emulsifiers, Solvents and / or propellants are processed (see H. Sucker et al: Pharmaceutical Technology, Thieme-Verlag, Stuttgart, 1978).
  • hydrophobic properties also enable transdermal Application of the new hirudin derivatives especially in connection with Penetration enhancers such as Dimethyl sulfoxide, alcohols or also azones or by iontophoresis.
  • the dosage depends on the age, condition and weight of the patient as well as on the type of application. Depending on the application form and Indication is usually the daily dose of active ingredient between about 20 to 40,000 ATU / kg body weight.
  • the hirudin conjugates can also be used successfully for antithrombogenic Coating artificial surfaces, such as Hemodialysis membranes and the necessary tube systems, used in vascular replacement or cardiopulmonary machines become.
  • the reaction mixture was washed with ice-cold 1N sulfuric acid, water and saturated sodium chloride solution and the organic phase was dried over magnesium sulfate.
  • the crude product was purified by fractional distillation. The main fraction was 317.3 g of a colorless liquid at a top temperature of 135 ° C and a pressure of 0.3 mbar. According to GC analysis, the product consists of 97% octyldiketene. IR, 1 H and 13 C NMR spectra agree with the proposed structure.
  • a hirudin mutein was used as the hirudin distinguishes natural hirudin HV1 by the following amino acid exchanges: Pos. 27: Lys; Pos. 33: Lys; Pos. 36: Arg; Pos. 47: Arg.
  • the production of such a hirudin mutein is described in DE 40 14 260 described.
  • hirudin mutein 20 mg were dissolved in 1 ml of 100 mM sodium borate buffer pH 9.0 dissolved and with 1 ml n-propanol and 1.5 mg octyldiketene added and then with intensive stirring at 4 ° C. incubated.
  • the hirudin-diketene complexes elute under the following conditions: % B 1 diketene unit per hirudin (octyl diketene 1 hirudin) 56% 2 diketene units per hirudin (octyl diketene 2 hirudin) 60% 3 diketene units per hirudin (octyl diketene 3 hirudin) 68%
  • the reaction mixture was washed with 1 N sulfuric acid, water and saturated saline and the organic phase was dried over magnesium sulfate.
  • the crude product was purified by fractional distillation.
  • the main fraction obtained is 172.8 g of a colorless liquid at a transition temperature of 95 to 100 ° C and a pressure of 0.15 mbar.
  • the product consists of 98% hexyldiketene.
  • IR, 1 H and 13 C NMR spectra agree with the proposed structure.
  • the solvent was distilled at 60 ° C and reduced pressure away. 75 g of sunflower fatty acid chloride were obtained as yellow oil with a chloride number of 12.0.
  • hirudin mutein 20 mg were in 1 ml of 100 mM Sodium carbonate pH 9.5 dissolved and with 1 ml of a solution of 1.5 mg palmitic acid N-hydroxy-succinimide ester (Sigma) in 1,4-Dioxane added and at 20 ° C with stirring for the reaction brought. After a reaction time of 4 h was added of a 5-fold molar excess of butylamine the reaction with stopped the hirudin (one hour at room temperature).
  • the palmitic acid 1- hirudin elutes at 57%, the palmitic acid 2- hirudin at 62% and the palmitic acid 3- hirudin at 76% solvent B.
  • the hirudin derivatives eluted at 100% B (oleic acid 1- hirudin and oleic acid 2 -hirudin) and 30% methanol (oleic acid 3 -hirudin).
  • the specific activities were 11000 U / mg for oleic acid 1- hirudin, 6200 U / mg for oleic acid 2- hirudin and 6600 U / mg for oleic acid 3- hirudin.
  • hirudin solution 20 mg / ml in 0.1 M Na carbonate or Na borate pH 9.5
  • a solution of 3.2 mg of N-hydroxysuccinimide activated cholesterol in 1.2 ml of THF and Incubated for 4 h at room temperature.
  • the reaction was by addition stopped by ethanolamine (2-fold molar excess based on Cholesterol) and the pH adjusted to 7.0.
  • the cholesterol-hirudin eluted at 38 minutes.
  • the specific Activity of the derivative was 16000 U / mg.
  • the activated cholesterol was produced as follows:
  • the farnesyl alcohol p-nitrophenyl carbonate was prepared as follows:
  • hirudin solution 21 mg / ml in 0.1 M Na carbonate or Na borate pH 9
  • tetrahydrofuran 0.5 ml
  • Lutensol TO3-N-hydroxysuccinimide carbonate Example 12
  • the reaction was carried out by adding ethanolamine (2-fold molar excess related to activated Lutensol) and stopped Chromatography - as described in Example 10 - separated.
  • the hirudin-lutensol derivatives eluted at 45% B (derivative 1 11300 U / mg, for derivative 2 9700 U / mg and 440 U / mg for derivative 3.
  • the starting material was produced as follows:
  • a hirudin solution 21 mg / ml in 0.1 M Na carbonate or Na borate pH 9
  • a hirudin solution 21 mg / ml in 0.1 M Na carbonate or Na borate pH 9
  • 4 mg of N- (2-hexyl-3-oxo-decanoyl) phenylalanine-N-hydroxysuccinimide ester added and 12 h at room temperature with stirring incubated.
  • the reaction was started by adding ethanolamine (2 times molar excess based on activated phenylalanine) stopped and by RP-HPLC - as described in Example 10 - separated.
  • the hirudin diketene derivatives eluted at 43% B (Derivative 1), 48% B (derivative 2) and 58% B (derivative 3).
  • the specific activities were determined as in Example 2 and were 1 930 U / mg for derivative, 2800 U / mg for derivative 2 and 135 U / mg for derivative 3.
  • the starting material was produced as follows:
  • 1.0 ml of a hirudin solution (21 mg / ml in 0.1 M Na carbonate or Na borate pH 9) was diluted with 1.0 ml of tetrahydrofuran and with 7 mg N- (6- (2-hexyl-3-oxo-decanoylamino) hexanoyloxy) succinimide added and incubated for 12 h at room temperature with stirring.
  • the reaction was carried out by adding ethanolamine (2-fold molar excess based on activated diketene) and stopped by RP chromatography - as described in Example 10 - separated.
  • the hirudin diketene derivatives eluted at 48% B (derivative 1), 52% B (derivative 2) and 63% B (derivative 3).
  • the specific Activities were determined as in Example 2 and were for Derivative 1 7500 U / mg, for derivative 2 3400 U / mg and 80 U / mg for Derivative 3.
  • the starting material was produced as follows:
  • Hirudin conjugates or placebo were anesthetized Rats or awake dogs administered intravenously. To defined At times, animals were given venous blood samples to collect Taken citrate plasma. The free anti-factor IIa activity of the Hirudin conjugates in plasma were determined using a chromogenic assay determined from a standard curve with recombinant hirudin.
  • Fresh citrate blood (9 parts blood + 1 part sodium citrate 0.11 mol / l, rat: 8.5 + 1.5) is centrifuged for 16 min at 250 xg to obtain platelet-rich plasma (PRP, supernatant).
  • PRP platelet-rich plasma
  • the PRP is used according to a method by Patscheke et al. (Haemostasis 10, 14-27, 1981) obtained a concentrate of washed platelets.
  • the platelets are first sedimented from the PRP by centrifugation at 330 xg for 7 min.
  • hirudin derivatives If treatment with hirudin derivatives is to take place, 200 ⁇ l of the derivative are added in a washing solution (120 mmol / l NaCl, 5 mmol / 1 KCl, 2 mmol / l CaCl 2 , 1 mmol / l MgCl 2) at this point , 5 mmol / l glucose, 2 g / l albumin, 50 mg / l apyrase in 30 mmol / l sodium phosphate, pH 6.5) for a final concentration of the derivative of 0.215 mg / l swirled the pellet and incubated for 10 min at room temperature.
  • a washing solution 120 mmol / l NaCl, 5 mmol / 1 KCl, 2 mmol / l CaCl 2 , 1 mmol / l MgCl
  • the platelet aggregation is determined by changing the measured transmission per unit of time in the sample (slope method). As a measure of the relative effectiveness and affinity of the hirudin derivatives, the thrombin concentration in NIH units / ml is determined as EC50, with which the half-maximum increase in platelet aggregation is achieved.
  • the neutralization index (NI) with NI EC50 derivative / EC50 control is calculated from this EC50 and the EC50 of a sample preincubated without hirudin. This value is derivative-specific and indicates the factor by which the thrombin concentration as agonist must be increased in order to again achieve the same aggregation (half-maximum slope) as in the control.
  • NI Control placebo-treated
  • NI Control placebo-treated
  • r-hirudin 1.21
  • Stearoyl hirudin 5.42
  • Palmitoyl hirudin 4.71
  • Cholesteryl hirudin 21.2
  • Lutensol T03 hirudin 15.1
  • the free ends of the catheters are covered by a 20.0 mm long glass capillary (inner diameter 1.0 mm) connected, which acts as a thrombogenic surface can i.v., s.c., p.o. or as an infusion.
  • a 20.0 mm long glass capillary inner diameter 1.0 mm
  • the shunt is passed through the test substance or solvent (control) Remove the clamps open.
  • the blood flow through the shunt leads to a rapid increase in the shunt temperature that occurs at the Middle of the glass capillary is measured.
  • the increase in room temperature on body temperature is an indicator of patency of the shunt.
  • the temperature is up to the closure of the Shunts recorded continuously for a maximum of 30 minutes.
  • the ED15min is calculated as the dose received on the control group to increase the shutter speed leads by 15 minutes.
  • the shunt and at the end of the Experiments will also use blood samples to determine anti-FIIa activity taken in plasma.
  • thrombus is triggered by a constant current flow (3 mA, 1 min) through a hook-shaped pair of electrodes, 1 cm distal to the flow measuring head on the surface of the vessel is applied.
  • Vascular occlusion is defined as one Reduction of the volume flow to ⁇ 0.3 ml / min for more than three minutes.
  • the antithrombotic effectiveness of hirudin derivatives is quantified as the frequency of thrombosis within 30 min in a group of 10 animals.
  • the ED50 is called the Dose calculated based on the control group the thrombosis frequency reduced by 50%.

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EP95901432A 1993-12-02 1994-11-25 Hirudin-konjugate aus hirudin und lipophilen verbindungen Expired - Lifetime EP0730473B1 (de)

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DE4341115 1993-12-02
DE4341115 1993-12-02
PCT/EP1994/003901 WO1995015183A1 (de) 1993-12-02 1994-11-25 Hirudin-konjugate aus hirudin und lipophilen verbindungen

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DE4437604A1 (de) * 1994-10-21 1996-04-25 Basf Ag Konjugate aus einem Poly- oder Oligopeptid und einer niedermolekularen lipophilen Verbindung
US5726168A (en) * 1995-10-12 1998-03-10 Eli Lilly And Company Lipophilic benzothiophenes
DE19915862A1 (de) * 1999-04-08 2000-10-12 Max Planck Gesellschaft Verwendung von molekulargewichtserweitertem Hirudin als Antikoagulans bei der extrakorporalen Nierenersatztherapie
AU2002363106B2 (en) * 2001-10-24 2008-04-24 Power Paper Ltd. Device and method for controlled delivery of active substance into the skin
US7438925B2 (en) * 2002-08-26 2008-10-21 Biovention Holdings Ltd. Drug eluting coatings for medical implants
BR102017005783A2 (pt) * 2017-03-21 2018-10-02 Fund Butantan processo de obtenção de esculptina e seus fragmentos, esculptina recombinante e seus usos
KR102366360B1 (ko) * 2021-06-29 2022-02-23 (주)네오팜 신규한 유사 세라마이드 화합물 및 이를 포함하는 피부 외용제 조성물

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0077529B1 (de) * 1981-10-16 1985-07-17 Sanol Schwarz GmbH Arzneimittelformulierung
DE3445532A1 (de) * 1984-12-13 1986-06-19 Plantorgan Werk Heinrich G.E. Christensen, KG, 2903 Bad Zwischenahn Hirudin-pa, desulfatohirudine-pa, verfahren zur herstellung und pharmazeutische mittel, die diese wirkstoffe enthalten
US4939174A (en) * 1988-02-26 1990-07-03 Shashoua Victor E Appetite suppression with dopamine-fatty acid conjugates
DE3819079A1 (de) * 1988-06-04 1989-12-07 Hoechst Ag Hirudin-derivate mit verzoegerter wirkung
WO1990010448A2 (en) * 1989-03-07 1990-09-20 Genentech, Inc. Covalent conjugates of lipid and oligonucleotide
EP0502962B1 (de) * 1989-12-01 1996-03-13 BASF Aktiengesellschaft Hirudin-Muteine und deren Polyalkylenglykolkonjugate
DE4014260A1 (de) * 1989-12-01 1991-06-06 Basf Ag Hirudinpolyalkylenglykolkonjugate
ATE131471T1 (de) * 1989-12-22 1995-12-15 Commw Scient Ind Res Org An fette gebundene aminosäuren, peptide oder deren derivate
AU7759291A (en) * 1990-03-29 1991-10-21 Gilead Sciences, Inc. Oligonucleotide-transport agent disulfide conjugates
US5256641A (en) * 1990-11-01 1993-10-26 State Of Oregon Covalent polar lipid-peptide conjugates for immunological targeting
WO1992019233A2 (en) * 1991-04-29 1992-11-12 Control Delivery Systems, Inc. Sustained release composition and methods of use thereof
EP0541737B1 (fr) * 1991-05-02 1997-04-09 BAUDET, Pierre Les n-phenyl-cinnamamides, protecteurs contre les effets nocifs de la lumiere ultra-violette
US5359030A (en) * 1993-05-10 1994-10-25 Protein Delivery, Inc. Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same

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AU679811B2 (en) 1997-07-10
DE4437502A1 (de) 1995-06-08
NZ276410A (en) 1997-03-24
JPH09505810A (ja) 1997-06-10
ZA949562B (en) 1996-06-03
NO962261D0 (no) 1996-05-31
US5919762A (en) 1999-07-06
SK67596A3 (en) 1996-12-04
CN1142776A (zh) 1997-02-12
NO962261L (no) 1996-05-31
ES2163487T3 (es) 2002-02-01
CZ157096A3 (en) 1996-09-11
IL111811A0 (en) 1995-01-24
SG49156A1 (en) 1998-05-18
CA2176967A1 (en) 1995-06-08
GR3036938T3 (en) 2002-01-31
HRP940968A2 (en) 1997-08-31
WO1995015183A1 (de) 1995-06-08
PT730473E (pt) 2002-02-28
BR9408195A (pt) 1997-08-26
FI962299A0 (fi) 1996-05-31
EP0730473A1 (de) 1996-09-11
FI962299A (fi) 1996-05-31
DK0730473T3 (da) 2001-10-08
HUT74390A (en) 1996-12-30
ATE204763T1 (de) 2001-09-15
DE59409847D1 (de) 2001-10-04
HU9601471D0 (en) 1996-07-29
PL314797A1 (en) 1996-09-30
AU1067095A (en) 1995-06-19

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